Analysis of Hox10 specific peptide motifs in their patterning functions of the axial skeleton

Tese de mestrado, Biologia (Biologia Evolutiva e do Desenvolvimento), 2009, Universidade de Lisboa, Faculdade de CiênciasHox genes play a fundamental role in anterior-posterior patterning and are remarkably conserved throughout evolution (Slack et al., 1993). Their products are transcription factors that regulate a specific set of genes with essential functions in development. Although different Hox genes show a notable functional specificity in vivo, they demonstrate a surprisingly low DNA-binding specificity in vitro. Sequence analysis can provide a way to understand how Hox genes achieve their biological specificity (Prince, 2002). Genetic experiments revealed that Hox genes are involved in global patterning processes in the axial skeleton to produce the axial formulae. Hox group 10 genes, in particular, have been shown to repress thoracic rib formation, since their overexpression in the presomitic mesoderm causes a ribless phenotype and their global inactivation resulted in extra ribs (Wellik et al., 2003, Carapuço et al., 2005). Two peptide domains were identified in Hox10 proteins which are conserved among all the Hox 10 members and are absent from all other Hox proteins. One of these is an octapeptide located just N-terminal to the homeodomain. The purpose of this work is to understand the role of this octapeptide in Hox10 protein function. This is being approached by the genesis and functional analysis of transgenic mice expressing mutant Hoxa10 proteins that contain specific deletions or amino acid changes in this domain. In previous transgenic assays, the overexpression of Hoxb9 gene in the presomitic mesoderm did not produce an abnormal axial skeleton phenotype. For this reason, this gene was used to generate chimeric contructs with the Hoxa10 gene. The results obtained show that the removal of the octapeptide is sufficient to block the rib-repressing activity of Hoxa10 when expressed in the presomitic mesoderm. In addition, introduction of this peptide motif, as well as the whole Hoxa10 sequence N-terminal to it, into the Hoxb9 protein produced a partial ribless phenotype. These results indicate that the octapeptide is necessary for the rib-repressing activity of Hoxa10 but it does not seem to be sufficient for this function, at least individuallyResumo alargado em português disponível no document

A pendulum of induction between the epiblast and extra-embryonic endoderm supports post-implantation progression

Embryogenesis is supported by dynamic loops of cellular interactions. Here, we create a partial mouse embryo model to elucidate the principles of epiblast (Epi) and extra-embryonic endoderm co-development (XEn). We trigger naive mouse embryonic stem cells to form a blastocyst-stage niche of Epi-like cells and XEn-like cells (3D, hydrogel free and serum free). Once established, these two lineages autonomously progress in minimal medium to form an inner pro-amniotic-like cavity surrounded by polarized Epi-like cells covered with visceral endoderm (VE)-like cells. The progression occurs through reciprocal inductions by which the Epi supports the primitive endoderm (PrE) to produce a basal lamina that subsequently regulates Epi polarization and/or cavitation, which, in return, channels the transcriptomic progression to VE. This VE then contributes to Epi bifurcation into anterior- and posterior-like states. Similarly, boosting the formation of PrE-like cells within blastoids supports developmental progression. We argue that self-organization can arise from lineage bifurcation followed by a pendulum of induction that propagates over time

A pendulum of induction between the epiblast and extra-embryonic endoderm supports post-implantation progression

Embryogenesis is supported by dynamic loops of cellular interactions. Here, we create a partial mouse embryo model to elucidate the principles of epiblast (Epi) and extra-embryonic endoderm co-development (XEn). We trigger naive mouse embryonic stem cells to form a blastocyst-stage niche of Epi-like cells and XEn-like cells (3D, hydrogel free and serum free). Once established, these two lineages autonomously progress in minimal medium to form an inner pro-amniotic-like cavity surrounded by polarized Epi-like cells covered with visceral endoderm (VE)-like cells. The progression occurs through reciprocal inductions by which the Epi supports the primitive endoderm (PrE) to produce a basal lamina that subsequently regulates Epi polarization and/or cavitation, which, in return, channels the transcriptomic progression to VE. This VE then contributes to Epi bifurcation into anterior- and posterior-like states. Similarly, boosting the formation of PrE-like cells within blastoids supports developmental progression. We argue that self-organization can arise from lineage bifurcation followed by a pendulum of induction that propagates over time